A major limitation in the development of non-viral gene carriers for Cystic Fibrosis (CF) has been the sparse attention paid to effects of the mucosal barrier on stability of gene carriers and on their ability to access and efficiently enter, traffic within, and deliver cargo DNA to the nucleus of lung epithelial cells. Absorption of mucus components often destabilizes gene carriers and significantly changes important carrier physicochemical properties that affect gene delivery efficiency. However, modifications aimed at overcoming one barrier (e.g. mucin absorption) may create other significant barriers (e.g. reduced cell entry rate). Our overall hypothesis is that a more comprehensive and quantitative "systems" approach to the identification of barriers to successful gene delivery for CF will allow our rational synthesis of novel gene carriers that resist mucus absorption and are capable of: (i) rapid transport through the mucosal barrier;(ii) facile entry into human CF bronchial epithelial cells following incubation in CF mucus;(iii) efficient and active accumulation around and in the cell nucleus within minutes of cell entry;and (iv) significantly improved gene expression in cell culture and in CF mouse models with gene carriers that have been pre-incubated in CF mucus. Specifically, starting with the synthesis and characterization of novel highly compacted (<22 nm in minor diameter) polymeric gene carriers (Aim 1), this proposal will utilize a number of powerful biophysical techniques to identify and quantify the rate limiting barriers to efficient gene carrier transport through human CF mucus (Aim 2) and through the cell to the nucleus (Aim 3). To ensure clinical relevance, gene carrier transport will be investigated in purulent/infected sputum and differentiated primary human bronchial epithelial cells grown at an air-interface, each freshly obtained from CF patients. Promising carriers will be tested for efficacy in a CF mouse model (Aim 4).The identification of important barriers in Aims 2-4 will guide the rational modification of the gene carrier physicochemical properties and surface chemistries (Aim 1 again) to potentially overcome the bottleneck. An interdisciplinary team, with expertise in bioengineering/biophysics, aerosol gene delivery, cellular trafficking/biology, and CF, has been assembled to investigate the hypothesis, with a long-term goal of safe and effective CF gene therapy.